The research objective is to elucidate the molecular mechanism by which calcium-dependent changes in actin filament length regulates the defense functions of lung macrophages, the contractility of smooth muscles, and the clearance of actin filaments from the blood stream. Particular emphasis will be focused on a 91,000 dalton, calcium-binding protein first identified and isolated by me from rabbit lung macrophages, and subsequently found to exist in a wide variety of nonmuscle and muscle cells, as well as extracellularly in blood plasma. This protein, called gelsolin, fragments actin filaments in the presense of micromolar concentrations of ionized calcium, preventing the formation of actin gel networks. Its action is reversible, and through regulation of gel-sol transformation of the cytoplasm, gelsolin is likely to be a key control point for many cytological events. In the first aspect of this proposal, research will be directed at examining the relation between the structure and function of gelsolin. Gelsolin will be subjected to limited proteolysis to identify the part of the molecule involved in binding to actin. The basis for activation of gelsolin by CA++ will be explored and the interaction between gelsolin and actin will be characterized. It is hoped that these studies will provide a better understanding of the molecular mechanism by which gelsolin shortens actin filaments. The second aspect of this work will be to explore how gelsolin, found in high concentration in smooth muscle, may play a role in the CA++=dependent regulation of smooth muscle contraction. Attempts will be made at localizing this protein intracellularly by immunofluorescent and immunoelectron microscopy. The effect of antigelsolin antibody on the contractility of smooth muscle cell models will be studied. A third aspect of this work will explore the hypothesis that gelsolin, present at a substantial concentration in plasma, reduces the length of actin filaments accidentally released into the blood stream, and thereby removes them as potential threat to the microcirculatory system. The structure and origin of plasma gelsolin will be determined, and its interaction with actin in blood examined. The possibility that gelsolin concentration changes during disease states will be explored.